It is proposed to analyze the DNA replication of the bacteriophage phi X174 under the electron microscope, using site-specific markers as reference points. The DNA of closely related phi X-like phages which form a characteristic heteroduplex molecule with phi X174 DNA, will be used to map the cistrons at the origin of DNA replication and at the strand termini in single-strand DNA replicating intermediates. E. coli ribosomes that bind specifically to phi X174 cistron G, and E. coli polymerase that binds specifically to promoter sites will be used (in lieu of partial denaturation that is not possible in phi X174) as fixed reference points to map the mode of replication of phi X174 double- stranded DNA. The methods will be applied to studying the DNA replication of tumor associated Parvoviruses, H-1 and minute mouse virus. A crude in vitro lysed cell system of phi X174 single-stranded DNA synthesis will be adapted for in vitro complementation of extracts of cells infected with phi X174 mutants defective in viral single-stranded DNA synthesis. This system will be used as an assay to isolate the phi X174 protein involved in single-stranded DNA synthesis,particularly the cistron C protein. A purified in vitro system of phi X174 single- stranded DNA synthesis will be constructed using these proteins and the molecular events involved in the initiation of single-stranded DNA synthesis on a double-stranded circular template will be investigated.